The blood brain barrier (BBB) is composed of brain microvessel endothelial cells (referred to hereafter as BMECs) and acts as a regulatory interface for permeability of drugs and solutes between the blood and central nervous system (CNS). Isolation and culture of BMECs have led to the development of in vitro model systems for studying the BBB (Bowman et. al, 1983, Audus and Borchardt, 1986).
In vitro BBB model systems have been successfully derived from bovine, canine, human, murine, porcine, and rat brains and were found to have similar permeability properties due to similarity of the physiological characteristics of the BBB among all mammals (Cserr et al, 1984, Audus et al, 1990). In these models, BMECs retain the characteristics of brain endothelial cells in vivo including morphology, specific BBB enzyme markers, and tight intercellular junctions which can be useful for studying a variety of CNS drug delivery issues ranging from passive diffusion, carrier mediated transport, and metabolism to specific factors affecting the BBB permeability. However, passaging of BMECs results in loss of specific endothelial and BBB markers as well as tight intercellular junctions (Brightman and Neuwelt, 1989).
Presently, only primary cultures of BMECs have been developed to address the BBB permeability issues in vitro. Isolated and cultured primary brain cells developed previously have exhibited different properties primarily due to considerable variety in the starting material. For example, with respect to transcellular transport, rigorous comparison of data between different laboratories has been very difficult (Pardridge et al., 1990, Masereeuw et al., 1994). Passaging primary cells can affect the differentiation of cells and lead to the selection of the most rapidly proliferating clones. Furthermore, the expression of some marker enzymes such as gamma-glutmayl transpeptidase as well as tight junctional complexity has been shown to decrease with time in culture and passage number (Meresse et. al., 1989).
Papoviruses such as SV-40 and polyoma virus are known to produce tumors in rodents. The polyoma virus DNA sequence has been determined and the `early region` which includes the small, middle and large T antigens has been indicated to be responsible for tumor formation. See Soeda et al., 1980, incorporated herein by reference. Previous methods using the SV 40 large T antigen gene disclose immortalization of bovine brain capillary endothelial cells in which the cells retain the differentiated phenotype and appear morphologically normal (Durieu-Trautmann et al., 1991). Fetal rat stems cells transfected with the middle T antigen gene produced two cell lines of endothelial cells that were not oncogenic, however, these cell lines do not spontaneously form a barrier to small hydrophilic molecules (Juillerat-Jeanneret et al., 1992).
Thus, it is apparent that the presently available clones of immortalized BMEC cultures suffer from individual drawbacks in terms of phenotype expression and homogeneic maintenance of that expression. This leads to difficulties with respect to accuracy and reproducibility in studies utilizing BMECs.